Fluorescence microscopy plays a major role as a diagnostic tool in many natural-science disciplines. The fundamental principle of fluorescence microscopy is to irradiate a sample with short-wave excitation radiation whereupon the sample itself, or a fluorescing dye with which the sample is stained, emits longer-wave fluorescent light (primary or secondary fluorescence) upon excitation with the short-wave excitation radiation. For fluorescence microscopy, secondary fluorescence is generally used to visualize certain specimen structures of stained preparations. Using fluorescence microscopy it is possible, for example, to identify pathogens, localize genes or determine genetic changes in DNA that is being examined, or visualize protein formation in cells.
A particular examination method using specific fluorescing dyes (so-called fluorochromes) is available depending on the application. Excitation using UV light for the “DAPI” dye, blue light for the “FITC” dye, or green light for the “Texas Red” or “rhodamine” dyes is, for example typical. Typical excitation frequencies are in the ultraviolet and visible spectral region.
Short-arc lamps filled with mercury or xenon, or halogen lamps, are normally used as light sources in the illumination systems for fluorescence microscopes. The light sources are most frequently located in a separate lamp housing that is adapted to the microscope. The aforementioned light sources possess a substantially continuous spectrum (UV to IR) that is interspersed with characteristic lines of high intensity. The spectral region appropriate for excitation of a fluorochrome can be selected from the spectral region of the light source by means of various (exchangeable) dielectric filters, called excitation filters. The bandwidth of the excitation filters is typically approximately 10 to 30 nm.
It is typical to work with different fluorescence filter systems (so-called filter blocks or filter cubes) so that different stains in a preparation can be visualized. These fluorescence filter systems comprise a mutually coordinated combination of an excitation filter, a dichroic splitter, and a blocking filter. The dichroic splitter reflects the excitation radiation to the preparation, but is transparent to the fluorescent light emitted from the preparation. The blocking filter shields the preparation from scattered excitation light that enters the objective. It possesses very high transparency, however, for the specific fluorescent radiation. The various fluorescence filters are usually located on a changing device that is embodied, for example, as a slider or carousel. Operation occurs manually and/or in motorized fashion.
Disadvantages of the aforesaid lamps of hitherto usual fluorescent illumination systems are low efficiency (since of the approximately 100 W of electrical power, only a few milliwatts reach the sample in the selected spectral region) and the troublesome heat evolution resulting therefrom. Lastly, the service life of the lamps is limited to a few hundred hours.
Very recently, light-emitting diodes or LEDs have been proposed and offered on the market as light sources for fluorescence microscopes.
DE 20 2004 010 121 U1, for example, describes a light source for an incident-light fluorescence microscope that comprises a high-power LED that emits blue light (460 to 480 nm). An arrangement having a single LED does not, however, meet the usual requirements of a routine or research laboratory.
An arrangement for fluorescent illumination is described in the Japanese application having the publication number JP 2005-321453; here the light of a light-emitting diode is conveyed via a collector lens into a fluorescence filter system and from there onto a specimen. The fluorescent light emitted from the specimen is directed via a blocking filter onto a CCD detector and/or an eyepiece. Also, this arrangement with a single LED cannot meet the requirements imposed here. The corresponding EP 1 593 996 A2 describes an expansion of this system to two light-emitting diodes, whose light fluxes are combined via a dichroic splitter and conveyed to a fluorescence filter system.
A fluorescence microscope in which lasers are used as light sources is described in the Japanese application having the publication number JP 07-333516. Here the light of two lasers is directed via dichroic splitters into a common illumination beam path. The fluorescent light emitted from the specimen is likewise conveyed through the same dichroic splitter to corresponding photodetectors. With this device, the specimen can be illuminated simultaneously with two excitation wavelengths, and the two resulting fluorescence wavelengths can be detected separately. A sequential excitation with different wavelengths is not possible when the lasers are operated continuously.
DE 103 14 125 B4 describes an LED light source arrangement for specimen illumination for fluorescence-microscopy applications, in which arrangement multiple diodes are arranged on a turntable; by rotation of the turntable about an axis at the center of the turntable, a specific diode can be selected and positioned in front of a light exit opening. Located at the light exit opening is a collimator optical system that couples the emitted light of the diode into the illumination beam path of the fluorescence microscope. Each light-emitting diode is joined to the turntable via a Peltier element to dissipate heat, and a retainer.
A disadvantage of this known LED light source arrangement is that changing to a different wavelength is associated with a mechanical movement of the turntable carrying the diodes. A change of wavelengths therefore takes a relatively long time and is associated with vibration and noise because of the mechanical movement. Because the diodes require good cooling, Peltier elements and/or correspondingly large masses are necessary in the receiving apparatus. In addition, electrical leads to the LEDs and to the Peltier elements on the turntable are problematic (wiper contacts or limited rotation range).
Lastly, the company COOLLED offers a light source in which multiple diodes are coupled in parallel in a liquid light guide. The three colors can be adjusted independently as to their intensity. Disadvantages of this illumination system are principally its size and weight. The liquid light guide furthermore generates light losses and light fluctuations, and requires an additional adapter to permit connection to usual lamp interfaces of the microscopes.
From DE 196 24 087 A1 furthermore an illumination apparatus for generating light having a high beam power level and large bandwidth is described. For this purpose, multiple single-point light sources, such as light-emitting diodes, that each emit light in a preferred direction, are spatially arranged with respect to one another by means of a holding device in such a way that the single-point light sources emit their light in at least two different directions; an optical device in the form of a reflector is provided, which device modifies the characteristics of the light emitted from the single-point light sources in such a way that it directs the radiated light in a predetermined direction. If the light sources have different wavelengths, a homogeneous color mixture of the light having different wavelengths can be achieved by corresponding configuration of the reflector as a diffuser. The conformation of the illumination apparatus proposed therein can resemble that of a usual halogen radiator. In an embodiment, the light emitted from multiple light-emitting diodes strikes a reflector, by which it is directed homogeneously onto the exit opening of the reflector and additionally color-mixed and collimated, so that the viewer can no longer detect the fact that the light derives originally from individual light-emitting diodes. The specific problems and requirements of an illumination device for fluorescence-microscopy examinations are not discussed in this document.
The long service life (several thousand hours) of LEDs, their rapid operability with no need for warm-up time, and the stable radiation power output, sufficient for fluorescence excitation, in a specific wavelength region having full widths at half maximum of approximately 20 to 50 nm, are advantages that make LED-based illumination systems for fluorescence microscopes superior to conventional systems.